Effect Of Gene Y On Biological Phenotype Of Oral Squamous Cell Carcinoma Cells And Its Mechanism

Part One Establishment of gene Y in oral squamous cell lines with high expression and knockdown stability and its influence on biological characteristics Objective:By changing the expression of gene Y in oral squamous cell carcinoma cells,the effects of gene Y on the biological behaviors such as proliferation and migration of the cell were studied,so as to explore the role of gene Y in the occurrence and development of oral squamous cell carcinoma,providing clues and directions for exploring its molecular mechanism and searching for tumor markers.Methods:1.Lentivirus particles with high expression and knockdown gene Y were transfected into oral squamous cell carcinoma cells(scc-25 cells),and the stable cell line was established after purinomycin(puro)screening.QPCR assay and Western Blot assay were used to verify the expression of gene Y at mRNA and protein level in the stable cell line.2.Real-time unmarked cell function analyzer was used to determine the growth curve to detect the effect of gene Y on cell proliferation ability,transwell assay was used to detect the effect of gene Y on cell migration and invasion,and clone formation was used to detect the effect of gene Y on cell migration and invasion.The effect of gene Y oncell proliferation was detected,and the effect of gene Y on cell cycle distribution was detected by flow cytometry.3.The effect of gene Y on the growth of subcutaneous tumor in nude mice was detected by subcutaneous tumorigenesis test in nude mice.Result:1.QPCR and Western Blot confirmed that the mRNA and protein levels of gene Y in the cells were significantly increased and decreased,indicating that the stable transfected cell line with over expression and knock-down of gene Y in oral squamous cell carcinoma were established successful.2.Cell growth experiments showed that high expression of gene Y could promote the growth rate of oral squamous cell carcinoma cells,and knockdown gene Y could inhibit the growth rate of oral squamous cell carcinoma cells.3.Cloning formation experiments showed that high expression of gene Y could promote the proliferation ability of oral squamous cell cells,and knockdown gene Y could inhibit the proliferation ability of oral squamous cell cells4.Transwell test showed that high expression of gene Y could promote the migration and invasion ability of oral squamous cell carcinoma cells,and knockdown gene Y could inhibit the migration and invasion ability of oral squamous cell carcinoma cells5.Flow cytometry results showed that high expression of gene Y could promote mitotic activity of oral squamous cell carcinoma cells and affect cell cycle distribution;Knockdown gene Y can inhibit the mitotic activity of oral squamous cell carcinoma cells and affect the distribution of cell cycle.6.The results of subcutaneous tumorigenesis in nude mice showed that knockdown gene Y inhibited subcutaneous tumorigenesis in nude mice.Conclusion:1.Highly expressed gene Y affects the cell cycle distribution of oral squamous cell carcinoma cells,enhances cell proliferation,speeds up cell growth,and promotes cellmigration and invasion.2.Knockdown gene Y affects cell cycle distribution of oral squamous cell carcinoma cells,weakens cell proliferation ability,slows down cell growth rate,and inhibits cell migration and invasion ability.Part two Regulation of Gene Y on the expression of different splicing variants of Gene U and Construction of stable Transformation Cell Line of Gene U with different splicing variants.Objective:By changing the expression of different splicing variants of gene U in oral squamous cell carcinoma cells,we studied whether gene Y regulated the expression of different splicing variants of gene U,so as to explore whether gene Y was involved in tumorigenesis by regulating the expression of different splicing variants of gene U.Methods:1.Lentivirus particles with different splicing variants of gene U were transfected into oral squamous cell carcinoma cells,and stable cell lines were established after purinomycin screening.2.Agarose gel electrophoresis and DNA polyacrylamide gel electrophoresis(DNA-PAGE)were performed with the products of qPCR.The target bands we needed were found under ultraviolet light observation,and the DNA were cut down and purified.The purified DNA was sequenced and verified.Results:1.The results of DNA polyacrylamide gel electrophoresis(DNA-PAGE)showed thatgene Y regulated the expression of different splicing variants of gene U.2.The results of agarose gel electrophoresis showed that the stable transfected cell line of U(15),U(15-)and NC control were successfully established in scc-25 cells.Conclusion:1.Gene Y plays a regulatory role in the expression of different splicing variants of gene U.After high expression of gene Y,the expression content of U(15+)increases while that of U(15-)decreases.After knocking down gene Y,U(15+)expression was decreased,while U(15-)expression was increased.2.The stable cell lines of different splicing variants of gene U were successfully established.